Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid
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molecules Article Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes Luboš Fical 1 , Maria Khalikova 1, Hana Koˇcová Vlˇcková 1, Ivona Lhotská 1 , Zuzana Hadysová 1, Ivan Vokˇrál 2 , Lukáš Cervenˇ ý 2 , František Švec 1 and Lucie Nováková 1,* 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; fi[email protected] (L.F.); [email protected] (M.K.); [email protected] (H.K.V.); [email protected] (I.L.); [email protected] (Z.H.); [email protected] (F.Š.) 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; [email protected] (I.V.); [email protected] (L.C.)ˇ * Correspondence: [email protected]; Tel.: +420-495-067-304 Abstract: Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, Citation: Fical, L.; Khalikova, M.; LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction KoˇcováVlˇcková,H.; Lhotská, I.; in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt Hadysová, Z.; Vokˇrál,I.; Cervený,ˇ L.; medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane Švec, F.; Nováková, L. Determination transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome of Antiviral Drugs and Their using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover Metabolites Using Micro-Solid Phase the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and Extraction and UHPLC-MS/MS in hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of Reversed-Phase and Hydrophilic stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using Interaction Chromatography Modes. Molecules 2021, 26, 2123. https:// BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile doi.org/10.3390/molecules26082123 and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) Academic Editor: James Barker conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited Received: 22 March 2021 superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity Accepted: 5 April 2021 of two orders of magnitude for many compounds. Method validation results indicated that HILIC Published: 7 April 2021 mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes Publisher’s Note: MDPI stays neutral leading to signal enhancement in RP and signal suppression in HILIC. with regard to jurisdictional claims in published maps and institutional affil- Keywords: UHPLC-MS/MS; hydrophilic interaction chromatography; reversed phase; antiviral iations. drug; microextraction; solid phase extraction Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Antiviral drugs are an important class of compounds because many viruses can This article is an open access article distributed under the terms and cause life-threatening diseases, as we are now witnessing with the COVID-19 pandemic. conditions of the Creative Commons Antiviral drugs can act against viruses at different stages of the viral life cycle, including (i) Attribution (CC BY) license (https:// inhibitors of virion fusion or entry, (ii) inhibitors of uncoating, (iii) inhibitors of integrase, creativecommons.org/licenses/by/ (iv) inhibitors of nucleic acid synthesis, (v) protease inhibitors, and (vi) neuraminidase 4.0/). inhibitors [1]. Most antiviral drugs affect the viral synthesis step, thus, important groups Molecules 2021, 26, 2123. https://doi.org/10.3390/molecules26082123 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 2123 2 of 22 involve the reverse transcriptase inhibitors and the protease inhibitors, acting in the viral replication and maturation step, respectively. A variety of structurally different antiviral drugs has been developed to target these different stages and also different types of viruses, including generally four major groups of (i) herpes viruses, (ii) respiratory viruses, (iii) hepatitis A, B, and C viruses (HAV, HBV, HCV), and (iv) human immunodeficiency virus (HIV) [1,2]. To 2020, more than 100 antiviral drugs have been approved by US Food and Drug Administration (FDA) [2,3]. Most recently approved antiviral drugs belong among anti- HIV and anti-hepatitis C (HCV) drugs in addition to drugs combating newly emerging viruses, such as Ebola virus and SARS-CoV-2 [3,4]. As the development of a new antiviral drug is very time-consuming and costly, the strategy of approvement of drugs previously registered in different indications for the new treatment purpose called drug repurposing, is currently also adopted in antiviral drug development [5,6]. Well-established doses and regimens are known for these drugs, their side effects and prevention are defined, synthetic routes for their preparation were implemented, and safety and quality assurance achieved. Therefore, approval of a new indication is usually faster, easier, and less expensive [6]. To support the evaluation of both, new antiviral drugs and repurposed antiviral drugs, their quantitative analysis in biological materials is of key importance to determine bioavailability, drug metabolism, and other pharmacokinetic parameters, as well as to allow therapeutic drug monitoring in later stages. Oral intake of antiviral drugs is the most common route of drug administration because it is considered the safest, most convenient, and highly economical. However, absorption of the drug from the gut is significantly af- fected by the presence of the intestinal barrier functioning as a selective filter for xenobiotics, including drugs. This barrier contains a range of tools, including xenobiotic-metabolizing enzymes and efflux transporters that prevent xenobiotics from entering the systemic circu- lation [7]. Concomitant oral administration of multiple drugs that are substrates, inducers, or inhibitors of these enzymes and/or transporters can subsequently result in drug-drug interactions (DDI) and hence in altered plasma levels [7]. DDI can occur in both directions meaning failure of the therapy in case of decreased plasma level of the drug below the therapeutic level and toxic effect if this level is exceeded. The risk of DDI is more common in aging patients characterized by comorbidities and polypharmacy [8]. Moreover, combination therapy using several antivirals from different classes with different mechanisms of action and therapy optimization are common in complex HIV treatment and in some cases of HCV treatment [9]. Anti-HIV and anti-HCV drugs are the typical examples of drugs administered orally where a high risk of DDI on intestinal efflux transporters can be expected [10]. Regulatory authorities as FDA and European Medicines Agency (EMA) are aware of this risk. Therefore, they recommend cell line- based assays (e.g., Caco-2, MDCK) to reveal the DDI in preclinical research [11,12]. New, more complex promising models as precision-cut intestinal slices (PCIS) are also emerging in this process [10]. As anti-HIV and anti-HCV drugs are administered in combination therapy, preclinical research methods must be complex enough to reveal the majority of possible DDI in different experimental models. To support the antiviral therapy aspects discussed above, multianalyte analytical methods allowing to monitor a large spectrum of antivirals in a single analytical run with high sensitivity and selectivity are beneficial. Although it is unlikely that numerous antivirals can be simultaneously present in biological sample since typical combinations involve two to three antivirals, a unique multianalyte method that can separate them all in a single run is desirable. It would allow separation of any combination without carrying out often tedious optimization of a dedicated methods enabling switching among these combinations and testing different compounds for DDI. Therefore, multianalyte methods are more suitable than single or several-analyte methods. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) is the method of choice that meets all these requirements [1]. The bio- analytical methods used in the analysis of antiviral drugs were recently summarized in two comprehensive review papers we published that focused on individual groups of Molecules 2021, 26, 2123 3 of 22 antivirals [1,9] and covered the period of 2000–2017. In addition, the most recent review article described the antivirals against COVID-19 [13]. The importance of multianalyte bioanalytical methods [14–20] has been emphasized already in our above stated review papers. Various newly published research papers have also demonstrated successful use of LC-MS/MS